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On the role of Cys150 in the mechanism of maleamate amidohydrolase (NicF)
Author(s) -
Noel Jeff W.,
Spittle Nicholas E.,
Snider Mark J.
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.756.20
Subject(s) - amidohydrolase , chemistry , enzyme , biochemistry , hydrolase , stereochemistry , catabolism , cysteine , mechanism (biology) , iodoacetamide , metabolic pathway , philosophy , epistemology
The oxidative degradation of nicotinate (vitamin B 3 ) to fumarate is considered a model system for the bacterial degradation of N‐heterocyclic compounds, yet only recently has the complete set of genes corresponding to the enzymes involved in this pathway been identified. The identification of these six genes has enabled structural and functional analyses of the enzymes of this catabolic pathway to be undertaken. Maleamate amidohydrolase (NicF), catalyzes the penultimate reaction of the pathway ‐ the hydrolytic deamination of maleamate to maleate. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The crystal structure offers insight into the roles of potential catalytically important residues, notably a conserved triad (Asp29, Lys117, Cys150) hypothesized to catalyze an addition‐elimination mechanism. In an effort to examine the functional importance of Cys150 in the mechanism iodoacetamide was discovered to be a potent inactivator. To test whether inhibition of NicF by sulfhydryl blocking reagents is reversible a study with methyl methanethiolsulfonate (MMTS) will be conducted. In addition, results of the kinetic analyses of NicF variants, Cys150Ser/Ala and Asp29Asn/Ala, are reported. This work was supported by a HHMI Undergraduate Science Education Program Award to the College of Wooster.